Upon binding of a retrovirus to a sensitive cell, the viral and cellular membranes fuse and the viral core is released into the cytoplasm. The viral genomic RNA is then reverse transcribed, yielding a double stranded cDNA copy of the viral RNA genome. A complex of viral DMA and proteins (the "preintegration complex" or "PIC") then carries out the covalent attachment of the viral cDNA to host DNA to form the integrated provirus. Each of these steps is required for viral replication. This proposal will utilize biochemical and fluorescence microscopy techniques to study the activity and composition of HIV PICs. The work will focus on purifying active PICs, those possessing the enzymatic activity and ability to carry out integration into an added target DNA in vitro. We are devising methods for combining PIC biochemistry with single particle imaging, which will provide a new window on PIC structure and function. We propose to 1) improve methods for isolating and purifying PICs, 2) improve methods for labeling PICs and determining their structure and composition, and 3) develop methods for in vitro and in vivo imaging of infected cells using labeled PICs. These proposed studies should greatly enhance our understanding of early stages of HIV infection and potentially contribute to the development of small molecule inhibitors of HIV integration.